BoNTs are a dangerous bioterrorism threat due to their extreme potency and lethality, as well as their ease of production and transport. If untreated, poisoning by the BoNTs can progress to flaccid paralysis and death due to respiratory failure. However, timely post-exposure intervention can limit the effects of the circulating toxin. The overall, long-term research objective is to generate a novel class of therapeutics that can be administered to individuals who have been poisoned by BoNT. The strategy, as described in this application, will be to develop dominant-negative mutants of one form of the toxin, BoNT/A, which will interact with and inactivate complexes of wild type toxin. BoNT/A is composed of two defined fragments. The H chain facilitates the transport of a second toxin fragment, the L chain, into target cells. The strategy is based on the hypothesis that transport of the BoNT/A L chain, which is an essential step in the cellular intoxication mechanism, is mediated by pH-triggered unfolding and membrane insertion of toxin oligomeric complexes. Experimental and computational approaches will be used to develop a model for the mechanism of H chain-mediated membrane transport of the L chain. This model will be tested by altering the amino acid sequence of the H chain in a manner predicted to interfere with membrane transport. Specifically, intramolecular disulfide linkages will be engineered within the H chain to limit movement of the polypeptide backbone as a result of pH triggered conformational changes. Moreover, positively charged amino acids will be introduced throughout the H chain, which would be predicted to be unfavorable for membrane insertion of the BoNT/A H chain. Wild type and mutant forms of the H chain will be expressed as recombinant proteins, and each mutant will be tested for dominant-negative inhibitory activity in the presence of wild type toxin. Simultaneously, structure-function relationships important for translocation will be identified as an important prerequisite for fu1ure design of dominant-negative based inhibitors. A milestone of this work will be the identification of one or more dominant-negative BoNT/A mutants that will block action of wild type toxin using in vitro model systems. The results from this research will establish the groundwork and justification for future development and in vivo testing of these novel inhibitors using established animal models.